Long-term Repopulation Research Articles

SALL4 is a robust stimulator for the expansion of hematopoietic stem cells

AbstractHSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by >10 000-fold for both CD34+/CD38− and CD34+/CD38+ cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34+ cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

Hematopoietic Stem Cells Survive Circulation Arrest and Reconstitute Hematopoiesis in Myeloablated Mice

Hematopoietic stem and progenitor cells (HSPC) for bone marrow transplantation are currently obtained directly from living voluntary donors or from cord blood units. However, a suitable donor is not always found. Because HSPC are known for their relative resistance to hypoxia, using an experimental murine model, we explored cadaveric bone marrow (BM) as their alternative source. After donor mice were sacrificed, BM was left in intact femurs at 37°C, 20°C, or 4°C under ischemic conditions, resulting in combined oxygen and metabolic substrate shortage and the accumulation of metabolic waste products. BM cells were harvested after a set time period ranging from 0 to 48 hours. To determine the impact of delayed harvesting on the transplantability of HSPC, a competitive repopulation assay using a murine Ly5.1/Ly5.2 congenic model in 2 different settings was used: after submyeloablative (6 Gy) or myeloablative (9 Gy) total-body irradiation, Ly5.2 hosts received cadaveric Ly5.1 cells or a mixture of cadaveric Ly5.1 cells and fresh Ly5.2 cells in a 1:1 ratio. Chimerism resulting from cadaveric donor cells, followed up to 6 months after transplantation, proved that the long-term repopulation ability of HSPC was fully preserved for 2 hours, 6 hours, and 12 hours at 37°C, 20°C, and 4°C of ischemia, respectively. A colony-forming unit-spleen (CFU-S) clonogenic assay revealed a higher sensitivity of proliferating hematopoietic progenitors to ischemia compared to repopulating cells (STRC and LTRC). Flow cytometry analysis of apoptosis in cadaveric BM demonstrated that the LSK (Lin(low)Sca-1(+)c-Kit(+)) subpopulation, enriched in HSPC, contained less apoptotic and dead cells than the BM as a whole. Furthermore, the number of LSK SLAM (CD150(+)CD48(-)) and LSK SP (side population) cells (fractions highly enriched in hematopoietic stem cells) decreased in parallel with BM transplantability. As well as cadaveric BM cells, we also tested the transplantability and survival of BM cells after storage in a suspension in vitro without specific hematopoietic growth factors. HSPC did not display any decrease in transplantability after 2 days of storage at 37°C or 4 days at 4°C. A higher sensitivity of progenitors to unfavorable conditions was observed again using CFU-S and granulocyte macrophage-colony forming cell (GM-CFC) assays, especially at 37°C. This paper shows that HSPC survive the cessation of circulation for a considerable time and maintain their engraftment potential. This time is significantly extended with in vitro storage compared to the cadaveric BM.

TIMP-1 deficiency subverts cell-cycle dynamics in murine long-term HSCs

AbstractIn addition to the well-recognized role in extracellular matrix remodeling, the tissue inhibitor of metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biologic functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of HSCs, whose biologic behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased BM cellularity and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− stem cells appears distorted, with a dysregulation at the level of the G1 phase. TIMP-1−/− HSCs also display increased levels of p57, p21, and p53, suggesting that TIMP-1 could be intrinsically involved in the regulation of HSC cycling dynamics. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish a role for TIMP-1 in regulating HSC function, suggesting a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu.

Critical role for Gimap5 in the survival of mouse hematopoietic stem and progenitor cells

Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1-which is essential for HSC survival-and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.

Abstract 1113: TGFβ pathway activation limits hematopoietic recovery from chemotherapy

Abstract Hematopoietic stem cells (HSCs) are capable of self-renewal and govern the long-term repopulation of all cell types of the blooding forming system. During normal hematopoiesis, there is a tight balance maintained between HSC self-renewal and differentiation. This balance is partially regulated through cell cycle regulators such as cyclin-dependent-kinases (CDKs) and CDK inhibitors (CKDIs); crucial positive and negative regulators of cell-cycle progression. We previously identified the CDKI p57Kip2 (Cdkn1c, p57) as an early transcriptional target of the tumor suppressor TGFβ that is required for TGFβ-induced cytostasis of human CD34+ hematopoietic progenitor/stem cells; functions that provide a mechanistic basis for its expression being silenced in many aggressive human myeloid and lymphoid malignancies. P57 is essential during late embryogenesis, functioning in tissue-specific developmental programs to coordinate proliferation, differentiation and apoptosis. We, and others, have found that p57 is highly expressed in deeply quiescent HSCs yet no role for p57 in hematopoiesis has been assigned. Utilizing a murine p57 knockout (KO) model, we studied the function of p57 in steady-state and stress hematopoiesis. Using limiting-dilution and competitive repopulation HSC assays, we found fewer long-term HSCs in the fetal liver of p57-KO animals demonstrating a role for p57 in HSCs development. In adult hematopoiesis, we found that p57 restrains the hematopoietic response to stresses imposed by serial-HSCs transplantation, hemolysis or myelotoxic chemotherapy. As a consequence, animal with p57-KO hematopoiesis tolerate higher doses of chemotherapeutics due to their more robust recovery from chemotherapy-induced myelosuppression. Using phospho-specific SMAD2 antibodies we found that the TGFβ pathway is activated in immature hematopoietic cells, in vivo, following chemotherapy-induced hematopoietic stress. Normally this promotes p57-induced cytostasis but p57-KO HSCs escape this restraint. We used 9-color flow cytometry to investigate how p57 affects hematopoietic recovery in long-term HSCs (LKSCD34- Flk2-), short-term HSCs (LKSCD34+ Flk2-), multipotent progenitors (LKSCD34+ Flk2+), common myeloid progenitors (LKSca-CD34+ FcRlo), granulocyte-monocyte progenitors (LKSca- CD34+ FcR+) and megakaryocyte erythroid progenitors (LKSca- CD34- FcR-). These results show that the most deeply quiescent HSCs undergo more robust self-renewal during stress in the absence of p57. Our results suggest that myelotoxic chemotherapy blockade of TGFβ-induced cytostasis, normally mediated by p57, could be used to limit chemotherapy-induced myelosuppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1113. doi:10.1158/1538-7445.AM2011-1113

Hoxb4-YFP reporter mouse model: a novel tool for tracking HSC development and studying the role of Hoxb4 in hematopoiesis

AbstractHoxb4 overexpression promotes dramatic expansion of bone marrow (BM) hematopoietic stem cells (HSCs) without leukemic transformation and induces development of definitive HSCs from early embryonic yolk sac and differentiating embryonic stem cells. Knockout studies of Hoxb4 showed little effect on hematopoiesis, but interpretation of these results is obscured by the lack of direct evidence that Hoxb4 is expressed in HSCs and possible compensatory effects of other (Hox) genes. To evaluate accurately the pattern of Hoxb4 expression and to gain a better understanding of the physiologic role of Hoxb4 in the hemato-poietic system, we generated a knock-in Hoxb4–yellow fluorescent protein (YFP) reporter mouse model. We show that BM Lin−Sca1+c-Kit+ cells express Hoxb4-YFP and demonstrate functionally in the long-term repopulation assay that definitive HSCs express Hoxb4. Similarly, aorta-gonad-mesonephrous–derived CD45+CD144+ cells, enriched for HSCs, express Hoxb4. Furthermore, yolk sac and placental HSC populations express Hoxb4. Unexpectedly, Hoxb4 expression in the fetal liver HSCs is lower than in the BM, reaching negligible levels in some HSCs, suggesting an insignificant role of Hoxb4 in expansion of fetal liver HSCs. Hoxb4 expression therefore would not appear to correlate with the cycling status of fetal liver HSCs, although highly proliferative HSCs from young BM show strong Hoxb4 expression.

Deletion of Puma and p21Waf1 In Mice Deactivates p53-Induced Cell Death and Cell Cycle Arrest, but Protects Mice From Irradiation-Induced Lymphomagenesis by a Mechanism Involving Hemopoietic Stem Cell Quiescence

AbstractAbstract 90 Introduction:The p53 gene is non-functional in >50% of human tumors. In mice deletion of p53 leads to a high incidence of tumors and to a significant acceleration of tumorigenesis induced by repeated gamma-irradiation. While a large number of effects have been described for p53, current concepts of p53-mediated tumor suppression discuss the roles of p53 in regulation of cell cycle and apoptosis as being essential. Two main targets have been identified in this respect: p21Waf1 as an essential regulator of cell cycle arrest downstream of p53 and Puma as the largest single contribution towards p53 induced cell death. Methods:We have generated p21Waf1/Puma doubly deficient (i.e. double-knockout – DKO) mice on a pure C57BL/6 background to investigate the effects on tumorigenesis. Results:In ex vivo irradiation studies DKO thymocytes expectedly showed reduced cell death and loss of a G1/S arrest upon irradiation. When following a cohort of mice for spontaneous tumor development, the DKO mice did not differ from wild-type (WT) controls. Since this may be explained by additional p53 down-stream effectors essential for tumor suppression, we set out to challenge the mice with an established repeated irradiation protocol (4 × 1.75 Gy over 4 weeks) in order to increase the likelihood of uncovering a defect in tumor suppression not apparent in unchallenged mice. While irradiated WT mice developed thymic lymphomas at an expected rate and p53 deficiency accelerated the lymphoma formation as published, irradiated DKO mice did not develop any thymic lymphoma at all. During the irradiation protocol WT mice followed a series of depletion and regrowth cycles in thymic cellularity with a high rate of cell death early post irradiations in TUNEL assays and a surge of proliferation on day 5 after irradiations detected by in vivo BrdU labeling. By contrast in DKO mice thymic cellularity dropped only slightly during the first irradiation cycle. This was followed by a slow and steady decline in cellularity over the following 3 cycles of irradiation. No late apoptotic wave or loss of proliferative capacity of remaining thymocytes could explain the loss of cellularity, nor could senescence of thymocytes be detected by SA-β-Gal staining in situ, suggesting that thymic influx was defective. It had previously been reported for the repeat-irradiation lymphomagenesis model, that the irradiation of hemopoietic precursor cells was essential for tumorigenesis. In contrast to thymic cellularity, DKO LSK numbers stayed relatively stable over the course of the 4 irradiations. By comparison WT LSK numbers dropped to about 50% by the time 4 irradiations were completed. Indeed, short-term repopulating (ST) cells dropped significantly, while long-term repopulating (LT) and multipotent progenitor (MPP) cell populations stayed more stable. In DKO marrows the relative content of LT, ST and MPP cells proved very stable across the irradiation schedule. In vivo BrdU labelling showed that WT LSK had a higher fraction of labelled cells at baseline and a >100% increase in the proliferative fraction during irradiation, while in DKO LSK the proliferation index was lower and stayed stably low over time, compatible with the replenishment defect observed in the thymus. DKO stem cells were only slightly more efficient (1.6-fold) than WT in bone marrow reconstitution experiments without challenge. However, when mixed chimeras were then subjected to the irradiation protocol with 4 × 1.75 Gy a clear advantage of the DKO cells became apparent (28-fold). Moreover, when reconstituting lethally irradiated mice with a mixture of WT and DKO marrow taken from repeatedly irradiated donors the efficacy ratio was 1:152. Conclusion:Our data contrast observations made in cell lines, where loss of Puma and p21Waf1 led to a p53-resistant outgrowth of cells. We present in an animal model that loss of Puma and p21Waf1 is not tumorigenic and in fact protects mice from irradiation carcinogenesis. Together with our recently published findings in irradiated Puma singly-deficient mice (Labi G&D 2010), our data suggest that tumorigenesis in irradiated DKO mice is inhibited by effects on hemopoietic stem cell reactivity to DNA damage. A combination of lack of generation of free niche space through protection of hemopoietic stem cells from cell death and a stem cell quiescence state retained in DKO stem cells after irradiation seems responsible for the phenotype. Disclosures:No relevant conflicts of interest to declare.

TIMP-1 Deficiency Subverts Cell Cycle Dynamics In HSCs.

AbstractAbstract 1545Although TIMP-1 was initially described as a mere inhibitor of Metalloproteinases (MMPs), recent findings have offered a different perspective on its biological role, contributing to unveil its multifaceted nature. In addition to inhibiting MMP activity, TIMP-1 has been proven to play MMP-independent, cytokine-like activities and to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. Bone marrow hematopoietic stem cells (HSCs) were isolated from TIMP-1-/- mice based on the phenotype Side Population c-Kit+Lin- Sca-1+ (SPKLS). In vitro cultural assays as well as in vivo transplantation assays were employed to investigate how TIMP-1 obliteration affects murine hematopoiesis. Cell-cycle dynamics in KO SPKLS HSCs were characterized by Pyronin Y/Hoechst staining, Ki-67 staining, as well as evaluation of RNA expression of cell cycle inhibitors, such as p53, p57, and p21.We found that TIMP-1-/- mice have decreased HSC numbers and, consistent with this finding, TIMP-1-/- HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1-/- LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Of note, TIMP-1-/- HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Interestingly, p53 RNA levels are indeed increased in TIMP-1-/- SPKLS HSCs compared to controls. Likewise, the expression level of other cell-cycle inhibitors, such as p57 and p21, were found to be higher in KO SPKLS HSCs, indicating a disregulation of cell-cycle dynamics.Our study highlights a novel biological role of TIMP-1 in the regulation of the HSC compartment and suggest a novel mechanism presiding over stem cell quiescence in the framework of the BM milieu. Disclosures:No relevant conflicts of interest to declare.

Circadian Corticosterone Levels Regulate and Integrate Hematopoietic Stem and Progenitor Cell Function and Bone Remodeling Via Notch1 Signaling

AbstractAbstract 840Hematopoietic stem and progenitor cell (HSPC) regulation involves the integration of signals emanating from the nervous system and various bone marrow (BM) cells. Stress signals are produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis via secretion of corticosterone (Cort in mice is the equivalent to cortisol in humans). The major stress hormone, Cort, is secreted in a circadian manner and is known to cause bone loss in supraphysiologic levels. However, whether Cort plays a role in regulation of HSPC and their interactions with the BM microenvironment is currently unknown. Moreover, potentially undesired effects of clinical glucocorticoid administration on HSPC function have not been investigated. We hypothesized that Cort acts in a circadian and dose dependent manner to orchestrate and integrate HSPC function and bone turnover, which are essential for host defense and homeostasis. To determine the roles of circadian Cort oscillations and effects of aberrant Cort regulation on HSPC biology, we utilized CRFR1-/- (R1KO) mice, which lack corticotropin releasing factor receptor 1, a known HPA axis receptor. These mice lack circadian Cort rhythms and have only residual plasma Cort levels. We found that R1KO mice have higher levels of hematopoietic progenitors and the more primitive SKL cells in their BM and blood compared to WT mice. Cort levels measured in the BM of R1KO mice were continuously low, contrasted to the normal and circadially oscillating levels detected in WT mice. As previously established, interactions of the chemokine SDF-1 (CXCL12), the pivotal regulator of stem cell migration and quiescence, with its major receptor CXCR4, are perturbed by various immune-mediated stress signals. Interestingly, the levels of SDF-1 were constantly high in the BM of R1KO mice with no typical circadian peaks. Furthermore, SKL cell release to the blood, expected to follow BM SDF-1-circadian oscillations, as previously shown by Frenette's group, was also constantly increased. Concomitantly, CXCR4 expression in R1KO SKL cells was reduced and the cells demonstrated higher proliferation rates with superior long-term repopulation capacity over WT transplanted cells. Rescue treatment of R1KO mice attained by adding Cort to their drinking water restored their BM SKL and SDF-1 levels to the basal values of WT mice. Increasing Cort levels continuously in the drinking water of WT mice diminished their BM phenotype, including long-term engraftment potential. In vitro cultures of normal BM cells yielded higher colony formation capacity when stimulated with low Cort levels (10-8M). Conversely, high Cort levels (10-5M) inhibited colony formation. Next, we examined microenvironmental components with HSPC support potential. R1KO mice had more primitive stromal cells (CD45-/CD11b-/CD29+/Sca-1+) in their BM. Expression of osteocalcin and osterix, genes characterizing mature osteoblasts, was reduced. Osteoclast maturation was also attenuated, as indicated by increased Gr-1-/CD11b-/c-Kit+ monocyte precursors and reduced transcription of the genes RANK and cathepsin K, both associated with mature osteoclasts and bone resorption. R1KO mice had fewer active osteoclasts in their bones and lower bone turnover rates as indicated by calcein/alizarin bone labeling. In addition, μCT analyses demonstrated a dramatic reduction in femoral trabecules and bone volume. Looking for mediating mechanisms, we found that R1KO BM mononuclear cells highly express Notch1 and its target gene Hes1, known to be involved in regulation of stem cell self renewal. Both genes reverted back to normal expression levels after adding Cort to the drinking water. The signaling Notch1 intracellular domain (NICD), a product of Notch1 cleavage by γ-secratase, was also upregulated in R1KO BM hematopoietic and stromal precursors. Finally, inhibition of Notch1 activation by adding the γ-secratase inhibitor DAPT to BM cultures prevented the stimulating effect of low Cort on colony formation, suggesting that this effect is mediated by Notch1 signaling. Taken together, our study suggests that abolishment of circadian Cort production coupled with continuous low Cort levels increases HSPC proliferation directly and indirectly via Notch1 signaling, while impairing bone maintenance and structure. We propose Cort oscillations and levels to be critical for integrating balanced bone turnover and HSPC function. Disclosures:No relevant conflicts of interest to declare.

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34(-)Flt3(-) KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34(-)Flt3(-) KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the modulation of angiocrine factors, with Akt-mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs.

Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells

AbstractDuring postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized microenvironments termed stem cell niches. Cell-cell and cell-extracellular matrix interactions between HSCs and their niches are critical for the maintenance of HSC properties. Here, we analyzed the function of N-cadherin in the regulation of the proliferation and long-term repopulation activity of hematopoietic stem/progenitor cells (HSPCs) by the transduction of N-cadherin shRNA. Inhibition of N-cadherin expression accelerated cell division in vitro and reduced the lodgment of donor HSPCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. Cotransduction of N-cadherin shRNA and a mutant N-cadherin that introduced the silent mutations to shRNA target sequences rescued the accelerated cell division and reconstitution phenotypes. In addition, the requirement of N-cadherin for HSPC engraftment appears to be niche specific, as shN-cad–transduced lineage−Sca-1+c-Kit+ cells successfully engrafted in spleen, which lacks an osteoblastic niche. These findings suggest that N-cad–mediated cell adhesion is functionally required for the establishment of hematopoiesis in the BM niche after BM transplantation.

HoxA cluster is haploinsufficient for activity of hematopoietic stem and progenitor cells

Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knockouts. Because HoxB genes are dispensable for hematopoiesis, and most HoxA genes are expressed an order of magnitude higher than other cluster genes in hematopoietic stem cell (HSC)-enriched populations, we hypothesize that maintenance of HoxA cluster expression is important for adult hematopoiesis and that global decrease of HoxA gene expression levels affects steady-state hematopoiesis. Expression levels of HoxA cluster genes have been determined in primitive hematopoietic populations derived from adult mice using quantitative reverse transcriptase polymerase chain reaction. Furthermore, the functional effect of single allelic deletion of the entire HoxA cluster on hematopoietic cells was analyzed by competitive repopulation assays using HoxA(+/-) mice. We show that the HoxA cluster is predominantly expressed in long-term HSCs and that expression declines with progression to short-term HSCs and early progenitors in a quantifiable manner. Monoallelic deletion of the HoxA cluster caused a general increase in primitive hematopoietic cell populations, but a decrease in side populations. In addition exhaustion of B-cell progenitors with age was observed, resulting in less mature B cells. Moreover, bone marrow of HoxA(+/-) mice had a significant larger population of Mac1/Gr1 neutrophils, which might be caused by accelerated maturation of myeloid progenitors. Transplantation assays demonstrated that HoxA(+/-) HSCs were less competitive in long-term repopulation of myeloablated recipients, which appeared intrinsic to HSCs. These results show for the first time that maintenance of adult HSCs and progenitors is particularly sensitive to HoxA gene levels, suggesting a specific role for the HoxA cluster in primary regulation of definitive hematopoiesis.

Abstract LB-266: Identification and enrichment of tumor-initiating cells from human prostate cancers using lentiviral vectors that can detect activated Wnt, Notch, or Hedgehog signaling

Abstract In the cancer stem cell hypothesis, tumors are initiated and maintained by a small subset of tumor initiating cells (TIC) that possess stem cell properties of self-renewal and long-term repopulation. However, TIC have not been well characterized in human prostate cancer with previous reports describing enrichment of TIC in prostate cancer based on expression of CD133 and a2b1 integrin cell surface markers. A significant problem has been the relative lack of cell surface markers that could be used to define subsets of prostate cancer cells. Rather than rely on cell surface markers, we wanted to identify subsets based on the presence or absence of the activated Wnt, Notch, and Sonic Hedgehog (Hh) signaling pathways that have been associated with stem cell maintenance and self-renewal, and also implicated in several cancers. We hypothesized that these signaling pathways may be more highly activated in TIC and may allow for the identification and enrichment of human prostate cancer TIC. To detect these signaling pathways, we generated lentiviral vectors with reporters downstream of specific enhancer-promoter sequences that can detect activated Wnt, Notch, or Hh signaling. Since established in vivo models that can recapitulate human prostate cancer from freshly isolated cancer cells do not exist, we developed in vitro clonogenic sphere formation assays, both in a 3-dimensional matrix and on non-adherent plates, to detect prostate cancer TIC. We first established the prostate cancer cells origin of the cells by demonstrating their invasive capacity in a matrigel cell invasion assay; their lack of CD10 and CD13 expression; and their ability to form non-adherent spheres when compared to normal prostate cells. The transduction efficiency of the prostate cancer cells by the lentiviral vectors was approximately 70%, with activated Wnt, Notch, and Hh signaling detected in 39%, 61%, and 34% of the transduced cells, respectively. We found that human prostate cancer cells with activated Wnt signaling produced approximately 10-fold more clonogenic spheres than Wnt-negative cells indicating enrichment of prostate cancer TIC within the Wnt-positive fraction. In contrast, prostate cancer cells lacking activated Notch signaling formed 2-fold more clonogenic spheres than cells with activated Notch signaling. The presence or absence of activated Hh signaling did not affect clonogenic sphere formation. When prostate cancer cells were transduced with both Wnt and Notch lentiviral vectors, the Wnt-postive/Notch-negative populations gave rise to 6-fold more clonogenic spheres than the Wnt-positive/Notch-positive cells. Finally, CD133+ and CD117+ prostate cancer cells were only detected within the Wnt-positive and Notch-negative populations. These results indicate that prostate cancer TIC are enriched within prostate cancer cells that have activated Wnt signaling but lack activated Notch signaling and suggest that prostate cancer subsets of interest can be identified based on the presence of activated signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-266.

Lin-/c-Kit+/Sca-1+造血前駆細胞の長期骨髄再建能を指標とした放射線感受性

Lin-/c-Kit+/Sca-1+造血前駆細胞の長期骨髄再建能を指標とした放射線感受性

The Earliest Stages of Loss of Stem Cell Self-Renewal in-Vivo Is Linked to Upregulated Biosynthesis of “Quiet” Mitochondria and Is Influenced by CXCR4 Activation and STAT3 Gene Deletion.

Abstract 2546Poster Board II-523One attribute of all stem cells is self-renewal (SR). Progress has been made in identifying genes/proteins involved in stem cell SR mechanisms, especially in embryonic stem cells, but very little is known in adult stem cells such as hematopoietic stem cells (HSCs). Mitochondria (Mt) have recently been considered more than an ATP generator and mediator of apoptosis. They act as a kind of scaffold for integrating numerous signals to and from the nucleus and from the extracellular environment, and signals involved in differentiation and cell cycle. They also stabilize microtubules of the mitotic spindle and suppress rotation of the mitotic division plane, potentially influencing asymmetric/symmetric stem cell divisions. Populations of primitive hematopoietic cells with low Mt activity (i.e. Rho123low) are highly enriched in long-term repopulating (LTR)-HSC. Because Mt can suppress mitotic spindle rotation by stabilizing spindle microtubules, potentially influencing asymmetric divisions, we hypothesized that Mt biogenesis/function could be important for mechanisms of SR loss and early-stage differentiation of mouse HSC. We examined Mt mass and Mt membrane potential in primitive mouse CD34lo/neg LINnegSca-1posc-kitpos (CD34-LSK) cells, CD34+LSK cells, and related populations. We note that as the small CD34-LSK cells begin to express CD34, Mt mass significantly increases (2×), while Mt membrane potential does not change in these CD34+LSK cells. The CD34+LSK cells then increase in size without further increases in Mt mass, but with upregulated Mt membrane potential. Because CD34 expression in LSK cells is associated with loss of LTR-HSC (i.e. self-renewal) and retention of only short-term repopulating ability, we propose this previously uncharacterized population of LSK cells with increased numbers/mass of “quiet” Mt may be an important component during initial stages of loss of SR and asymmetric HSC division. We found that primitive LSK cells from mice constitutively expressing an SDF-1α/CXCL12 transgene (TG mice) have shifted ratios of these various mitochondrially distinct populations that may be related to CXCR4 signaling and its known influence on glycolysis and STAT3 activity, both of which can influence Mt metabolism. LSK cells in TG mouse total bone marrow significantly increased from .039+/_.01% to .085+/_.02% (n=6), but they were predominantly CD34+LSK cells. These CD34+LSK cells were composed of two distinct types of cells; small/high-Mt mass and large/high-Mt mass cells. The small LSK cells have lower Mt membrane potential while the large LSK cells had increased Mt membrane potential. Because CXCR4 can signal through STAT3, and because STAT3 can bind to Mt and influence their metabolic activities (i.e. membrane potential), we hypothesized that mice with a tissue-specific targeted gene deletion of STAT3 in hematopoietic cells might result in LSK cells that are defective in upregulating Mt membrane potential, which could shift the proportions of the three mitochondrially distinct populations of LSK cells toward the smaller, Mt mass-low, Mt membrane potential-low type of LSK cell. Bone marrow from targeted STAT3-deleted had LSK numbers increased 50%, but they were composed almost entirely of small, Mt mass-high, membrane potential-low, CD34+ type of LSK cells. This is consistent with our hypothesis and suggests that Mt membrane potential may be important for latter stages of CD34-LSK cell differentiation to more lineage-committed progenitor cells but may not be required for initial stages of SR loss. We propose a model where Mt biogenesis occurs before/during loss of SR in CD34-LSK cells. These Mt are metabolically quiescent (“quiet”), attach to spindle microtubules of dividing cells, and influence spindle orientation resulting in loss of SR (small, Mt mass high, Mt potential low, CD34+LSK cells). Before further differentiation, these cells upregulate Mt metabolism/membrane potential and increase in size to facilitate further expansion and differentiation (large, Mt mass high, Mt potential high, CD34+LSK cells). This model could help explain the observed phenotypes of SDF-1 α/CXCL12 transgenic mice and hematopoietic cell-targeted STAT3 deleted mice. This information may also be useful in the search for strategies to prevent loss of SR capacity of HSCs in culture and expansion of HSCs in-vitro. Disclosures:No relevant conflicts of interest to declare.

High Resolution Purification and Characterization of Human Cord Blood-Derived CD34–negative SCID-Repopulating Cells with a Very Immature Phenotype.

Abstract 815We have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with an extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In that study, 13 lineage specific antibodies (Abs), including anti-CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41, CD56, and CD235a, were used to purify CD34− SRCs. A limiting dilution analysis demonstrated the frequency of CD34− SRCs in the 13 lineage-negative (Lin−) CD34− cells to be approximately 1/25,000. In this study, we added 5 more lineage specific Abs, including anti-CD11b, CD33, CD45RA, CD66c, and CD127, in order to highly purify CD34− SRCs. The 18 Lin−CD34− cells showed a homogeneously blast-like morphology, and their incidence in the CB-derived nucleated cells ranged from 0.0002 to 0.001%. The colony-forming capacity of these highly purified 18 Lin−CD34− cells was quite unique, since 50% of the total colony-forming cells (CFCs) were mixed colony-forming cells (CFU-Mix). In contrast, the 18 Lin−CD34+ cells formed myeloid, erythroid, and mixed colonies, however, only <10% of the total CFCs were CFU-Mix. The phenotypic and functional characterizations of these 18 Lin−CD34− cells were then further investigated by cocultures with the HESS-5 murine stromal cell line in the presence of a cocktail of cytokines, such as SCF, flt3 ligand, TPO, IL-3, IL-6, and G-CSF. After 7 days of coculture, the total number of cells was observed to expand by 20 to 30 folds, 40 to 60% of which were consisted of CD34+ cells. Next, we investigated the SRC activity of these 18 Lin−CD34− cells using NOD/Shi-scid mice. When 4×104 18 Lin−CD34− cells were transplanted using IBMI, all 4 mice were highly repopulated with human CD45+ cells, including CD19+ B-lymphoid and CD33+ myeloid cells. In addition, the level of human cell engraftment in the bone marrow (BM) ranged from 93.3 to 97.5% (median, 96.8%), at 12 weeks after the transplantation. Interestingly, the BM cells obtained from the primary engrafted NOD/ Shi-scid mice that received transplants of 1,000 to 2,000 18 Lin−CD34− cells showed a secondary repopulating capacity. Furthermore, a limiting dilution analysis demonstrated the frequency of CD34− SRCs in these 18 Lin−CD34− cells to be approximately 1/1,000. The next approach to characterize the CD34− SRCs with respect to the self-renewal potential as well as the long-term repopulating potential, was to serially analyze the kinetics of engraftment for 24 weeks in the NOD/Shi-scid/IL-2Rgcnull (NOG) mice that received transplants of 2,000 18 Lin−CD34− cells containing only 2 CD34− SRCs (estimated number). All 6 mice showed signs of human cell repopulation (0.4 to 28.9%, median 2.5%) at 5 weeks after the transplantation at the contra-lateral sites of IBMI. The repopulation rates gradually increased, and reached a high level of repopulation (47.0 to 87.9%, median 72.0%) at 18 weeks after the transplantation. These results indicated that CD34− SRCs could thus sustain a long-term human cell repopulation in NOG mice, thereby suggesting that CD34− SRCs are a distinct class of primitive HSCs in comparison to the previously reported CD34+ SRCs. Disclosures:No relevant conflicts of interest to declare.

CD61/ Integrin β3 Ligation Contributes to the Thrombopoietin-Mediated Niche Function of Mouse Hematopoietic Stem Cells.

Abstract 383Hematopoietic stem cells (HSCs) reside in a specialized microenvironment, bone marrow (BM) ‘niche' for maintaining self-renewal and multi-lineage differentiation capacities through the life. We recently reported that murine HSCs (CD34- c-Kit+ Sca-1+Lin-: CD34-KSL) but not CD34+KSL hematopoietic progenitors highly express CD61 (integrin β3), correlating well with long-term repopulating activity (LTRA) of HSCs (Umemoto et al., J. Immunol, 2006). However, the roles of CD61 on HSC regulation and underlying molecular mechanisms in the BM niche are to date unknown. We herein demonstrate a novel role of CD61 using CD61-deficient (CD61-/-) and mutant CD61 mice that exhibit 747 tyrosine residue substituted with alanine in the cytoplasmic tail of β3 integrin subunit (CD61Y747A KI) whereby the integrin inside-out and outside-in signaling are impaired. We found that the CD61-deficiency influences the sensitivity to 5-fluorouracil (5-FU) administration (0.25 mg/g i.v. one shot), increasing the mortality of CD61-/- mice (CD61-/- mice died within 14 days, n=20, vs. WT mice survive over 90 days, n=20, P<0.0001). HSCs per se appeared to be responsible for these results because CD61-/- CD34-KSL cells but not CD61-/- CD34+KSL cells showed accelerated cell-cycle progression compared to those of WT, suggesting that CD61 might contribute to maintenance of quiescent state of HSCs within the BM. It is known that thrombopoietin (TPO) acts on not only potent hematopoietic differentiation from HSCs but also for maintaining quiescent state of HSCs. Accordingly, we examined the relationship between TPO effect and integrin signaling through CD61 in HSCs. The in vitro single cell culture system showed the decreased cell proliferation and survival in CD61-/- CD34-KSL cells that were cultured in the presence of TPO (50 ng/mL), compared to the WT CD34-KSL cells (P<0.01). In contrast, CD61-deficency did not influence the phenotype of cultured CD34-KSL cells in the presence of stem cell factor (SCF, 50 ng/ml). Interestingly, these results were mirrored by CD61Y747A KI CD34-KSL cells and their littermate control CD34-KSL cells. Furthermore, when 40 CD34-KSL cells after 5 days culture in the presence of TPO and they were then applied into long-term competitive repopulation assay, CD61Y747A KI CD34-KSL cells showed less LTRA than that of WT (P<0.01). These results indicated that tyrosine at 747 of the β3 integrin subunit was required for the TPO-dependent expansion/maintenance of HSC in vitro. Whereas, to initiate integrin outside-in signaling in the presence of TPO, CD34-KSL cells were cultured on a well of 96-well plates pre-coated with various CD61 ligands (either vitronectin, 5 μg/ml or osteopontin, 5 μg/mL), resulting in the enhancement of their LTRA, compared to without these ligands. Since these ligands could interact with integrin receptors besides CD61, we utilized the antibody to CD61, 2C9.G2 that is capable of phosphorylating CD61 to mimic the actions of outside-in signaling. Using CD34-KSL cells were cultured with 2C9.G2, we further confirmed that CD61 signaling allowed CD34-KSL cells to maintain undivided state in vitro and increase LTRA in vivo, which were dependent upon the presence of TPO but neither SCF nor other growth factors including insulin growth factor-2 and fibroblast growth factor-1. More interestingly, both stimulation with CD61 ligation and TPO influenced ex vivo expansion of HSCs when started at 40 CD34-KSL cells in a 96-well culture well. Collectively, our results indicate that CD61/β3 integrin signaling plays important roles in TPO-mediated expansion/maintenance of HSC within the BM niche, which are possibly mediated through the tyrosine phosphorylation at 747 tyrosine residue of the β3 integrin cytoplasmic domain. Disclosures:No relevant conflicts of interest to declare.

Fractionated Osteoblastic Niche Cells Enhances the Homing Activity of Hematopoietic Stem Cells.

Abstract 560Hematopoietic stem cells (HSCs) retain quiescent state in the adult bone marrow (BM), interacting with specialized niches along the endosteum (osteoblastic niche) and in perivascular sites adjacent to endothelial and reticular cells (perivascular niche). We have previously reported that the regulations of HSCs in the osteoblastic niche through the receptor-cytokine, cell-to-cell, and cell-to-extracellular matrix (ECM) interactions are critical for the maintenance of cell cycle quiescence. However, osteoblast population is heterogeneous in terms of the degree of differentiation and function of each cell. In this study, we characterized the cellular components of osteoblastic niche and examined the ability for the maintenance of HSCs. We found that CD45–CD31–Ter119– endosteal population can be subdivided into three fractions, ALCAM+Sca-1–, ALCAM–Sca-1+, and ALCAM–Sca-1– cells. Expression of osteoblast markers and differentiation potential of these three fractions revealed that osteoblasts were enriched in the Sca-1– populations. On the other hand, ALCAM–Sca-1+ cells had characteristics of mesenchymal progenitor cells (MPCs). Microarray analysis showed that ALCAM+Sca-1– fraction tend to highly express the genes related to cell-to-cell or cell-to-ECM adhesions, such as N-cadherin, Osteopontin, and Alcam, compared to other fractions, indicating that this population might regulate HSCs through the physiological interactions. On the other hand, ALCAM–Sca-1+ fraction highly expressed the genes related to growth factor and cytokine that are involved in the regulation of both quiescence and proliferation of long-term-HSCs, such as Angiopoietin-1, Flt3l, Cxcl12, Thrombopoietin, and Kitl. These data rise the possibility that multiple cell populations in endosteum collaborate as a complex to regulate the balance between HSC proliferation and quiescence in the endosteum in adult BM. Next we examined whether these fractionated cells could maintain the long-term repopulation (LTR) activity of HSCs in vitro. BM Lin–Sca-1+Kit+ (LSK) cells were cocultured with each fractionated endosteal population without growth factors. After 2 days of coculture, Ly5.1+ cells were sorted and transplanted into lethally irradiated mice. We found that all three fractions maintained LTR-activity of LSK cells. In particular, LSK cells cocultured with ALCAM+Sca-1– cells showed significantly higher LTR-activity compared to that cocultured with other fractions. Then we analyzed the gene expression implicated in the homing and lodgment of HSCs. Q-PCR array analysis revealed that upregulation of Cxcr4, integrins, Cd44, N-cadherin and Alcam was induced in LSK cells cocultured with Sca-1– populations. In particular, ALCAM+Sca-1– cells significantly upregulated N-cadherin expression in HSCs. These data suggest that osteoblasts increased homing activity of HSCs in culture or enriched cell population that had high homing activity. Furthermore, the cell-to-cell adhesion between HSCs and ALCAM+Sca-1– cells enhanced the interaction of HSCs with niche complex and maintained self-renewal activity of HSCs. Disclosures:No relevant conflicts of interest to declare.

In Vivo divisional Tracking System Reveals Cycling Heterogeneity in Long-Term Hematopoietic Stem Cells.

Abstract 814Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and give rise to all mature cells of hemato-lymphoid system for the lifetime of an individual. To ensure this, HSCs are kept at homeostatic levels in adult bone marrow. Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assay using 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., PLoS ONE 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic model systems (Wilson et. al., Cell 2008; Foudi et. al., Nat. Biotech. 2008). Based on the latter, it was suggested that one HSC pool turns over faster than another, dormant pool with very limited divisions during a lifetime. However, the fast cycling HSCs did not have long-term multilineage reconstitution capacity in lethally irradiated animals in contrast to dormant HSCs (Wilson et. al., Cell 2008; Foudi et.al., Nat. Biotech. 2008). From these experiments remained unclear, whether the faster cycling HSC loose long-term repopulation potential according to divisional history, or whether they represent progenitors with limited self-renewal potential, sharing a long-term HSC phenotype. Therefore, the dynamics of steady-state long-term HSC homeostasis and blood production remains to be determined.To address this directly, we set up an in vivo HSC divisional tracking assay. Here we show i.v. transfer of CFSE (carboxyfluorescein diacetate succinimidyl ester) -labeled HSCs into non-conditioned CD45.1/2 congenic F1 recipient mice that allows evaluation of steady-state HSC dynamics as CFSE distributes equally to daughter cells upon each cellular division. Sorted naïve CD4+CD62L+ T cells were used as non-dividing control cell population to determine the zero division CFSE staining level over time. Upon transfer of Lin-c-kit+Sca-1+ cells (LKS) into sublethally irradiated mice, all donor derived Lin-c-kit+ cells had divided >5 times after 3 weeks. However, transfer of LKS cells into non-irradiated mice revealed non-divided LKS cells in recipient bone marrow over 20 weeks. FACS analysis with HSC or progenitor specific marker expression showed that most of 0-2 time-divided and few of >5x divided LKS cells maintained a long-term HSC phenotype (CD150+, c-mpl+, CD34-). In order to test HSC potential, non- or >5x divided cells were sorted based on divisional history from primary recipients at different time points after transplantation, and competitively transplanted into lethally irradiated secondary recipients. At 3 weeks post primary transfer, single non-divided LKS cell was able to multi-lineage repopulate recipients, while 50 of >5x divided LKS cells showed no engraftment. Interestingly, both non- and >5x divided LKS cells at 7 or 12-14 weeks after primary transfer had long-term multilineage repopulating potential. Limiting dilution transplantation experiments demonstrated that HSC with long-term multilineage capacity (LT-HSC) were maintained at constant numbers that fit the numbers of free bone marrow niche space, with non-divided LT-HSC decreasing and >5x divided LT-HSC increasing with a constant division rate. We next tested the effects of hemato-immunological challenge on HSC cycling dynamics. Upon i.p. LPS injection into mice, previously transplanted with CFSE-labeled LKS, almost all LT-HSCs entered cell cycle within one week after challenge.These findings directly demonstrate that some LT-HSCs are quiescent for up to one fifth of the life-time of a mouse, while other LT-HSCs divide more actively, thus proving asynchronous LT-HSC division and contribution to hematopoiesis in steady-state. In addition, the results demonstrate that quiescent LT-HSCs are driven into division in response to naturally-occurring hematopoietic challenges, such as systemic bacterial infection. The CFSE-tracking model established here now allows to directly test the role of intrinsic versus environmental cues on cycling-dynamics of HSCs as well as leukemia initiating cells in steady-state and upon challenge on multiple genetic and different species background. Disclosures:No relevant conflicts of interest to declare.

TIMP-1 Deficiency Subverts Cell Cycle Dynamics in Long-Term HSCs.

Abstract 2514Poster Board II-491In addition to the consolidated role in extracellular matrix remodeling, the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased HSC numbers and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Indeed, HSCs exhibit a higher proliferation rate, leading to an increased formation of CFU-C in vitro and spleen colonies (CFU-S) after transplant. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish TIMP-1 role in HSC function, suggesting a novel mechanism presiding over stem cell quiescence and potentially involved in the development of hematological diseases. Disclosures:No relevant conflicts of interest to declare.